In our previous work, we isolated a diploid epitherlial rat liver, hepatocyte cell culture and characterized its response to aflatoxin B1. We also succeeded in transforming this culture which demonstrated the transformation by aflatoxin B1, of a cell culture derived from the in vivo target tissue. In this project we propose to continue the characterization of the normal, toxin-transformed, and intrinsically transformed hepatocyte culture by morphological, karyological and biochemical analyses using cultures which had been sequentially stored in liquid nitrogen during the transformation process. We propose also to study the surprising differences in RNA biosynthesis between the diploid and heteroploid cultures used. We propose to study the interesting morphological variants found in long term normal cultures and in the ascites tumors derived from them. We shall also extend these studies to other carcinogenic hydrocarbons. We have also begun to develop a casein-overlay system which shows promise in its ability to detect putative carcinogens and to identify transformed epithelial cells in a culture of normal epithelial cells. Lastly, using hepatocyte cultures derived from human and primate sources, we will extend these studies to a more relevant system.